Continuous exposure to isoprenaline reduced myotube size by delaying myoblast differentiation and fusion through the NFAT-MEF2C signaling pathway

We aimed to explore whether superfluous sympathetic activity affects myoblast differentiation, fusion, and myofiber types using a continuous single-dose isoprenaline exposure model in vitro and to further confirm the role of distinct NFATs in ISO-mediated effects. Compared with delivery of single and interval single, continuous single-dose ISO most obviously diminished myotube size while postponing myoblast differentiation/fusion in a time- and dose-dependent pattern, accompanied by an apparent decrease in nuclear NFATc1/c2 levels and a slight increase in nuclear NFATc3/c4 levels. Overexpression of NFATc1 or NFATc2, particularly NFATc1, markedly abolished the inhibitory effects of ISO on myoblast differentiation/fusion, myotube size and Myh7 expression, which was attributed to a remarkable increase in the nuclear NFATc1/c2 levels and a reduction in the nuclear NFATc4 levels and the associated increase in the numbers of MyoG and MEF2C positive nuclei within more than 3 nuclei myotubes, especially in MEF2C. Moreover, knockdown of NFATc3 by shRNA did not alter the inhibitory effect of ISO on myoblast differentiation/fusion or myotube size but partially recovered the expression of Myh7, which was related to the slightly increased nuclear levels of NFATc1/c2, MyoG and MEF2C. Knockdown of NFATc4 by shRNA prominently increased the number of MyHC +, MyoG or MEF2C + myoblast cells with 1 ~ 2 nuclei, causing fewer numbers and smaller myotube sizes. However, NFATc4 knockdown further deteriorated the effects of ISO on myoblast fusion and myotube size, with more than 5 nuclei and Myh1/2/4 expression, which was associated with a decrease in nuclear NFATc2/c3 levels. Therefore, ISO inhibited myoblast differentiation/fusion and myotube size through the NFAT-MyoG-MEF2C signaling pathway.

Overexpression or knockdown of NFATs in vitro. Constructions of NFATc1/c2/c4 overexpression adenoviral vectors were prepared as previously described 17 . The gene accession numbers of overexpressing NFATc1/c2/c4 are NM_172390, NM_173091, and NM_004554, respectively. Constructions of NFATc3/c4 short hairpin RNA (shRNA) adenoviral vectors were prepared as previously described 17 . These overexpression adenoviral vectors containing Ad-NFATc1, Ad-NFATc2, Ad-shNFATc3 and Ad-shNFATc4 were obtained from Vigenebio. To confirm the role of NFATc3 or NFAtc4 in myoblast cells, Ad-shCtrl, Ad-shNFATc3, or Ad-shNFATc4 (1 × 10 9 pfu) was added to the corresponding culture dishes one day before ISO treatment. Then, these cells were replaced with differentiation medium for further observation. Myoblast differentiation. After myoblast cells were treated under DM for the indicated time, the differentiated myoblast cells were stained for MyoG or MEF2C through the first polyclonal antibody MyoG (sc-12732, 1:150, Santa Cruz) or MEF2C (5030S, 1:400, CST) and appropriative TRITC-labeled secondary antibody (Jackson Lab, 1:500, USA). The nuclei were marked by DAPI staining. The numbers of single-or double-positive nuclei in a high-power field (HPF, 50 μm) were analyzed after double staining with MyoG/DAPI or MEF2C/ DAPI. Images were evaluated by two people who did not know the results using ImageJ (Java) software (National Institutes of Health, USA). The percentage was calculated by the formula = MyoG-or MEF2C-positive nucleus numbers/DAPI-positive nucleus numbers.
To further distinguish the difference between differentiation and fusion, MyoG/DAPI-or MEF2C/DAPIpositive cells were divided into two types, and the corresponding cell numbers were evaluated. C2C12 myoblast cells with only 1-2 nuclei within a cellular structure were evaluated as analyzed by MyoG or MEF2C staining, indicating that MyoG + or MEF2C + cells were defined as differentiated cells without mutual fusion to myotubes. Myoblast cells with 3 and more than 3 nuclei in the structure of a cell were defined as myotubes. The numbers of double-positive nuclei in a high-power field (HPF, 50 μm) were analyzed after double staining with MyoG/ DAPI or MEF2C/DAPI. Images were evaluated by two people who did not know the results using ImageJ (Java) software (National Institutes of Health, USA).
Myoblast fusion and myotube morphology. The differentiated myoblast cells were stained for MyHC through the first polyclonal antibody MyHC (rabbit anti-mouse antibody, sc-20641, 1:150, Santa Cruz) and appropriate TRITC or FITC-labeled secondary antibody (Jackson Lab, 1:500, USA). C2C12 myoblast cells with only 1-2 nuclei within a cellular structure were evaluated as analyzed by MyHC staining, indicating that MyHC + cells were defined as differentiated cells without mutual fusion to myotubes. Myoblast cells with 3 and more than 3 nuclei in the structure of a cell were defined as myotubes. The nuclei were marked by DAPI staining.
To further analyze myotube size, the myotubes were divided into two types, and the myotube numbers, length and area were evaluated. One is a short myotube with 3 ~ 5 myoblast fusions; the other is a long myotube with more than 5 myoblast fusions. Morphology was assessed by myotube length, area (two types, less than 200 μm and more than 200 μm) and number of myotubes with myoblast fusion (3 ~ 5 nuclei or more than 5 nuclei) under high-power magnification 17,19 . Images were evaluated by two people who did not know the results using ImageJ (Java) software (National Institutes of Health, USA).
Quantitative RT-PCR. Using the SuperScript II cDNA kit (Invitrogen, Life Technologies), the total RNA extracted from C2C12 myoblast cells by TRIzol reagents (Invitrogen, Life Technologies) was transcribed into cDNA. Quantitative PCR was carried out by using SYBR green PCR master mix (Thermo Fisher Scientific, Applied Biosystems, CN) in a Real-Time PCR System (RotorGene 6000, Qiagen, Germany). The transcript levels of the gene of interest in each group were normalized to the GAPDH levels 20 . The primers used are listed in Table 1.
Western blot. C2C12 myoblast cells in the tubes were placed on ice and homogenized within 0.1% Tween-20 homogenization buffer with the addition of protease inhibitors. This was followed by the separation and collection of nuclear and cytosolic proteins from each group by using NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer's instructions (78,835, Thermo Fisher Scientific, USA). After electrophoresis in a 7 or 10% SDS-PAGE gel, 20 µg of protein from each group was transferred onto a PVDF membrane (Millipore). Then, the membrane was blocked with 5% nonfat milk. Subsequently, primary antibodies Statistical analysis. IBM SPSS statistics software (version 22, 32-bit edition) was used for for statistical analysis. https:// www. ibm. com/ cn-zh/ produ cts/ spss-stati stics. Data from quantitative and semi-quantitative analyses are presented as the mean ± SD. The paired or unpaired Student's t test was used to determine statistical significance between two groups. Comparison of results among more than three experimental groups should be made to specify their differences by one-way ANOVA. P < 0.05 was considered meaningful. IBM SPSS statistics, version 22, 32-bit edition.

Results
Continuous single-dose ISO most obviously hampered C2C12 myoblast differentiation and fusion. To confirm the distinct role of ISO on myoblast differentiation and fusion, we administered ISO at different frequencies, including once single-dose, interval single-dose or continuous single-dose, and detected the morphological changes of myotubes by MyHC immunostaining. As shown in Fig. 1, in differentiation medium containing 2% HS-DMEM, C2C12 myoblast cells time-dependently differentiated into mature muscle cells and formed myotubes characterized by MyHC-positive staining. Regardless of single-dose, interval single-dose or continuous single-dose delivery, there was no difference in myotube numbers with either 3-5 nuclei or more than 5 nuclei (5 + ) on day 2. However, on day 4, continuous single-dose ISO not only decreased the numbers of myotubes with 3-5 nuclei but also reduced the numbers of myotubes with 5 + nuclei, suggesting that continuous single-dose ISO hindered C2C12 myoblast differentiation and postponed myoblast fusion. Furthermore, on the fourth and sixth days of differentiation, there was almost a 50% inhibition ratio of myotube numbers with 5 + nuclei in response to continuous single-dose ISO compared to the one single-dose or interval single-dose ISO, indicating that continuous single ISO could stably inhibit myoblast fusion.
To further determine the dose-dependent effect of ISO with single-dose stimulation on C2C12 myoblast differentiation and fusion, 10 −8 , 10 −7 , 10 −6 or 10 −5 mol/L ISO was used to treat the cells once every day. As shown in Fig. 2, compared to the normal differentiation group, the number of MyHC-positive myotubes with either 3-5 or 5 + nuclei was evidently decreased with continuous single-dose ISO stimulation in a dose-dependent manner, especially 10 −5 mol/L ISO, reducing the number of 5 + nuclei myotubes by nearly two-thirds, indicating that continuous single-dose ISO inhibited C2C12 myoblast differentiation and fusion. Furthermore, in response to continuous single-dose ISO, the numbers of less than 200 μm myotubes were dose-dependently increased, while the numbers of more than 200 μm (200 + ) myotubes were decreased, especially 10 −5 mol/L ISO, diminishing the numbers of 200 + myotubes by nearly 50% compared to the normal differentiation group. Meanwhile, it increased the numbers of less than 200 μm myotubes by 3.5 times as much as the control group, indicating that continuous single-dose ISO substantially shortened the length of myotubes. In summary, continuous single-dose ISO diminished myotube size by hampering C2C12 myoblast differentiation and fusion.

Continuous ISO stimulation dose-dependently altered NFATc1 and NFATc2 signaling.
To further determine whether the dose-dependent effect of ISO with single-dose stimulation on C2C12 myoblast differentiation and fusion involved in NFAT signaling, different doses of ISO, including 10 −8 , 10 −7 , 10 −6 or 10 −5 mol/L, were continuously added to the medium containing 2% HS-DMEM. As shown in Fig. 3, continuous single-dose ISO dramatically reduced NFATc1 and NFATc2 in differentiating C2C12 myoblast cells in a dose- Table 1. The sequences of primers of qPCR.

Continuous ISO stimulation time-dependently altered NFATc1 and NFATc2 signaling.
To further determine the time-dependent involvement of NFAT signaling in C2C12 myoblast differentiation and fusion, western blotting was used to evaluate changes in protein levels at different times. As shown in Fig. 4A-E, in the normal differentiation group, there was a gradual increase in NFAT levels within 6 days. On day 4, the protein levels of NFATc1 ~ c4 were evidently increased compared to the levels on day 2 ( Fig. 4A-E). However, the protein levels on day 6 of differentiation were markedly decreased compared to those on day 4. These results indicated the dynamics of NFAT expression involved in myoblast differentiation and fusion. Following the stimulation of continuous single-dose ISO with 10 −5 mol/L, we found that NFATc1 levels in myoblast cells continuously decreased, while NFATc3 and NFATc4 levels increased. Similar to the normal differentiation group, NFATc2 presented an alternative pattern where there was a decrease and then an increase in levels during myoblast differentiation when stimulated with ISO. Regardless of a decrease or an increase, ISO treatment significantly influenced NFATc2 levels more than the control treatment ( Fig. 4A-E). These typical changes indicated that NFAT signaling could participate in the process of myoblast differentiation and fusion mediated by ISO.
NFAT signaling is involved in the regulation of myoblast differentiation/fusion and myotube size mediated by ISO. To determine the relationship between NFATs and ISO-mediated myoblast differentiation and fusion inhibition, adenovirus-mediated overexpression of NFATc1 and NFATc2 or knockdown of NFATc3 and NFATc4 by shRNA were used to observe the ISO-mediated effect on myoblast differentiation and fusion inhibition. First, His-tag, as an adenovirus vector-carried reporter gene, was detected, indicating that the adenovirus vector was successfully transfected with the results of the marked expression in myoblasts (Fig. 5A). Second, overexpressed NFATc1 or NFATc2 in myoblasts showed a 3-fourfold increase in mRNA levels (Fig. 5B,C), implying that NFATc1/c2 was successfully overexpressed in myoblasts. Knocking down NFATc3 www.nature.com/scientificreports/ and NFATc4 in myoblasts obliviously decreased the 70% mRNA levels (Fig. 5D,E), indicating that NFATc3/c4 were successfully knocked down in myoblasts. Finally, to confirm whether overexpression or knockdown of NFATs affects the expression and cytoplasm/nucleus distribution of other NFATs, we used western blot analysis. We found that overexpression of NFATc1 dramatically increased the nuclear NFATc1 and cytoplasmic NFATc2 levels while reducing the nuclear and cytoplasmic NFATc3/c4 and cytoplasmic NFATc1 levels (Fig. 5F,G). The difference was that overexpression of NFATc2 did not alter the cytoplasmic levels of NFATc3/c4 but obviously increased the nuclear NFATc1/c2 levels while lessening the NFATc3/c4 levels in nuclei (Fig. 5H,I). Of interest, while knockdown of NFATc3 by shRNA caused a decrease in nuclear and cytoplasmic NFATc3 levels, the nuclear levels of NFATc1/c2 were increased, especially NFATc1. Knockdown of NFATc4 by shRNA also increased the levels of nuclear NFATc1 but reduced the levels of nuclear NFATc2/c3 and cytoplasmic NFATc1/c2, accompanied by a decrease in nuclear and cytoplasmic NFATc4 levels ( Fig. 5F-I). These results indicated that NFAT interactions could be involved in the changes in myoblast differentiation and fusion induced by ISO. As per their respective functions, the overexpression of NFATc1/c2 abolished the suppressive effects of ISO on myoblast differentiation and myoblast fusion (Fig. 6). Moreover, overexpression of NFATc1 resulted in a more potent effect than overexpression of NFATc2 in restoring myoblast differentiation/fusion characterized by 5 + nuclei myotubes (Fig. 6A-C). Compared with the partial recovery of the ISO-mediated inhibition effect by overexpression of NFATc2, forced NFATc1 expression restored myotube length and area relative to the sizes in the normal differentiation group (Figs. 6A-C, 7A-B).
Of interest, Ad-shNFATc3 did not alter the ISO-mediated inhibition of myoblast differentiation/fusion compared with Ad-shNFATc4, worsening it. Although knockdown of NFATc4 by shRNA increased MyHC-positive cells with 1-2 nuclei in ISO-treated myoblast cells, it decreased the number of both 3-5 nuclei and 5 + nuclei myotubes ( Fig. 6A-C, Supplemental Fig. 1A), leading to a smaller myotube length and area (Figs. 1B and 7A-B), www.nature.com/scientificreports/ indicating that it did not change myoblast differentiation but did change myoblast fusion, which was associated with the decrease in NFATc2 and increase in NFATc1 induced by knocking down NFATc4 (Fig. 5E-H). Meanwhile, overexpression of NFATc4 partially restored the inhibitory role of ISO in myoblast fusion (Supplemental Fig. 2). Unlike knocking down NFATc4, knocking down NFATc3 did not alter the inhibitory role of ISO on myotube length and area, which was related to the increase in NFATc1/c2 induced by knocking down NFATc3 (Fig. 5E-H). Taken together, NFAT interactions could be involved in the inhibitory effect of ISO on myoblast differentiation/fusion and myotube length/size.

NFAT signaling is involved in alterations in myofiber marker genes mediated by ISO. Previous
studies have reported on two types of muscle fibers, including slow (slim-long) and fast (thick-short) myofibers. MyHC1 encoded by the Myh7 gene forms the former, and MyHC2a encoded by the Myh2 gene, MyHC2b encoded by the Myh4 gene or MyHC2X encoded by the Myh1 gene the latter 22,23 . Corresponding with decreased myotube formation following treatment with continuous single-dose ISO (Figs. 1, 2), the expression levels of Myh7, Myh4, Myh2 and Myh1 were significantly reduced (Fig. 7C-F). Moreover, there was a significant reduction in the expression of MyHC1, MyHC2a and MyHC2X compared with MyHC2b ( Fig. 7C-F)  subsequent processes of myoblast cell differentiation 24 , we used MyoG and MEF2C staining to confirm the relationship between NFATs and ISO-mediated myoblast differentiation. First, we observed whether specific overexpression or knockdown of NFATs affected the nuclear levels of MyoG and MEF2C. We found that the ratio of MyoG-or MEF2C-positive nuclei within the total nucleus was increased in these adenovirus treatment groups, resulting in increased numbers of myotubes ( Fig. 8A-C). The percentage of MyoG + nuclei was higher than that www.nature.com/scientificreports/ of MEF2C + nuclei in the differentiating C2C12 myoblast cells treated with these adenoviruses (Fig. 8A-C), indicating that MyoG/MEF2C are involved in myoblast differentiation and fusion mediated by NFATs. Subsequently, with continuous single-dose ISO treatment, the percentage of MyoG-and MEF2C-positive nuclei in C2C12 myoblast cells was markedly reduced (Fig. 9A,B). The percentage of MEF2C + nuclei was lower than that of MyoG + nuclei in ISO-treated C2C12 myoblast cells, indicating that ISO inhibited the initiation and subsequent processes of myoblast differentiation through MyoG and MEF2C, especially in MEF2C. Combined with the results that ISO did not significantly hamper the initial differentiation of myoblasts shown in Fig. 1, ISO mainly inhibited the anaphase of myoblast differentiation through MEF2C. More importantly, overexpressing NFATc1/c2 or knocking down NFATc3/c4 almost completely restored the number of MyoG-and MEF2Cpositive nuclei in ISO-treated myoblasts (Fig. 9A,B). Moreover, among these four adenoviruses, overexpressing NFATc1 restored the positive number of MyoG and MEF2C most strongly close to the normal level, leading to Myotube formation results from the fusion of differentiated myoblasts, characterized by more than three (3 + ) nuclei in the structure of a cell 17,19 . We found that continuous single-dose ISO markedly decreased the numbers of MyoG-and MEF2C-positive nuclei in 3 + myotubes, especially in MEF2C myotubes. Meanwhile, overexpression of NFATc1 almost completely restored the number of MyoG-or MEF2C-positive nuclei in 3 + myotubes in ISO-treated myoblasts, close to normal levels ( Fig. 9A-D). However, overexpression of NFATc2 in ISO-treated groups only partially recovered these changes. Similarly, knocking down NFATc3 partially recovered MyoG-or MEF2C-positive nuclei numbers in 3 + myotubes exposed to ISO. Interestingly, NFATc4 knockdown did not alter the reduced trends of MEF2C-positive nuclei in 3 + myotubes induced by ISO but partially recovered MyoG-positive numbers in 3 + myotubes (Fig. 9A-D). These results indicated that NFATs, NFATc1 in particular, participated in the suppressive role of continuous single-dose ISO in C2C12 myoblast fusion in coordination with MyoG and MEF2C.

Discussion
Due to long-term overactivity of the sympathetic nerve, injury induced by increased levels of NE and E often ascribes to β-AdR1 activation 7 . Typically, isoproterenol (ISO) has been used to imitate the role of β-AdR. This is because of its more stable binding property to β-AdR compared to that of NE and E to both α-AdR and β-AdR, which results in difficulty in analyzing AdR subtype functions 2 . In earlier studies, a single high dose of ISO (10 −4 M) was usually used to trigger skeletal muscle atrophy [25][26][27] . Herein, we first found that continuous single-dose ISO at a concentration below 10 −4 M evidently hindered C2C12 myoblast differentiation and fusion compared with single-dose or interval single-dose administration (Fig. 1A-B). Second, ISO administration with a continuous single dose reduced the nuclear levels of NFATc1/c2 and MyoG/MEF2C and increased the nuclear   (Fig. 10).  www.nature.com/scientificreports/ Continuous single-dose ISO has shown typical dose-dependent traits. However, at 10 −8 M and 10 −5 M ISO, myoblast differentiation/fusion and signaling molecules displayed obvious differences. For example, 10 −8 M ISO markedly reduced NFATc1/c2 levels (50-60%) in myoblast cells, and myoblast differentiation/fusion was slightly decreased at 10 −8 M ISO; however, these differences were not significant compared with the normal control group. Our previous studies have shown that compared with 10 −5 M ISO, 10 −8 M ISO did not alter pAKT, p38MAPK, or pERK1/2 levels, which play important roles in myoblast differentiation/fusion 9 . The increased pERK1/2 levels at 10 −5 M ISO inhibited myoblast differentiation and fusion through the inactivated AKT and activated FOXO1 signaling pathway. These specific effects could be obviously abolished by the ERK1/2 blocker PD98059, in line with Marino's report 28 , indicating that pERK1/2 levels at 10 −8 M ISO contributed to partial preservation of myoblast differentiation and fusion. Of interest, at 10 −5 M ISO, pERK1/2 is still at a high level, while nuclear NFATc1/c2 proteins are at a minimum level, leading to the remarkable inhibition of myoblast differentiation and fusion, consistent with the effect of increased nuclear FOXO1 and comparable inhibition of NFATs on pancreatic β cell dysfunction 29 .
Published data have shown that NFATc2 is an important player in the primary control of myoblast recruitment and myoblast fusion 19,30,32 . In addition to regulating the specialization of muscle fibers, NFATc1 could regulate myoblast fusion by promoting NFATc2 expression 48 , in line with our results that Ad-NFATc1 alone substantially increased the levels of NFATc2 (Fig. 5C-F), suggesting that the recovery of myoblast differentiation and fusion by overexpressing NFATc1 could be involved in the partial restoration of NFATc2 in ISO-treated myoblast cells. Of interest, overexpression of NFATc1 reversed the inhibitory effects of ISO on myoblast differentiation and fusion, resulting in increased myotube size, more than NFATc2 overexpression. This difference could be related to the fact that NFATc1 overexpression markedly increased the cytoplasmic and nuclear levels of NFATc2 accompanied www.nature.com/scientificreports/ by an obvious increase in the nuclear levels of NFATc1. Relatively, NFATc2 overexpression slightly increased the nuclear levels of NFATc1 with a significant increase in NFATc2 levels in the nucleus. Then, unlike the overexpression of NFATc1, NFATc2 overexpression apparently increased the cytoplasmic levels of NFATc3/4. Indeed, NFATc3 promoted myoblast differentiation and fusion, while NFATc4 inhibited it 35,[44][45][46][47] . We speculated that this high-level accumulation of cytoplasmic NFATc3/4 could be involved in the partial recovery effects of overexpressing NFATc2 on the length and area of myotubes. Although the detailed mechanism needs to be further clarified in the future, ISO inhibited myoblast differentiation/fusion, resulting in diminished myotube size, which was associated with the destroyed coordination of different members of the NFAT family. Regarding muscle fiber type specification, four NFATs are involved in the control of type I myofibers and slow muscle specialization [37][38][39][40][41][42][43] , especially NFATc1 37,38 . NFATc1-mediated slow muscle specialization and the fast to slow myofiber-type switch require MEF2C and MyoD coordination [40][41][42][43] , respectively. In this study, continuous single-dose ISO decreased the MEF2C levels within the nucleus, and the specific effects could be eliminated by overexpressing NFATc1 more than overexpressing NFATc2, resulting in stronger recovery of the inhibited type I muscle fiber, indicating that NFATc1 signaling controlled the changes in ISO-reduced type I muscle fiber, at least partly related to the aid of MEF2C. Of interest, in contrast to the results that the activated NFATc3 contributed to slow muscle marker gene Myh7 expression in the coordination of MyoD, knockdown of NFATc3 by shRNA partially recovered the expression of Myh7 in ISO-stimulated myoblasts cells, attributed to the compensatory changes caused by NFATc3 knockdown; that is, knockdown NFATc3 slightly increased the MEF2C levels of myotubes with more than 3 nuclei while raising the nuclear levels of NFATc1/c2. Meanwhile, knockdown NFATc4 decreased the expression of both Myh7 and Myh1/2/4, which was partially consistent with the result that NFATc4 mainly contributed to fast muscle fiber formation characterized by Myh1/2/4 30,38 . The other reasons for these changes could be related to the evidence that knockdown NFATc4 increased the nuclear NFATc1 levels while reducing the nuclear NFATc2/c3 levels, in addition to the fact that the MEF2C levels of myotubes with more than 3 nuclei were not changed. Therefore, ISO reduced type I and II myofibers, which could be associated with the destruction of NFAT synergy.

Conclusion
Our results provide a novel mechanism by which continuous single-dose ISO dramatically affects myoblast differentiation and fusion, myotube size and muscle fiber specialization through the NFAT-MyoG/MEF2C signaling pathway.